Acid Addition Salts, Hydrates and Polymorphs of 5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic Acid Ethylamide and Formulations Comprising These Forms

ABSTRACT

The present invention relates to new salt forms of 5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic acid ethylamide, in particular the mesylate, hydrochloride, tartrate, phosphate and hemi fumarate salt thereof; to crystalline forms of such salts; to polymorphs of 5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic acid ethylamide; to hydrates and polymorphs of the new salt forms mentioned above; to the use of the new salt forms mentioned above for the manufacture of a medicament for the treatment of a disorder mediated by Hsp90; to a method for treating a disorder mediated by Hsp90 using the new salt forms; to formulations comprising such salt forms, in particular aqueous solutions suitable for intravenous administration; and to amber glass containers being filled with such formulations.

The present invention relates to new salt forms and polymorphs of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide, to hydrates and polymorphs of such new salt forms andto new formulations comprising such salt forms.

The Hsp90 family of chaperones is comprised of four known members:Hsp90α and Hsp90β both in the cytosol, grp94 in the endoplasmicreticulum and trap-1 in the mitochondria. Hsp90 is an abundant cellularchaperone required for the ATP-dependent refolding of denatured or“unfolded” proteins and for the conformational maturation of a varietyof key proteins involved in the growth response of the cell toextracellular factors. These proteins, which are called client proteins,include the steroid receptors as well as various protein kinases. Hsp90is essential for eukaryotic cell survival and is overexpressed in manytumors. Cancer cells seem to be sensitive to transient inhibition ofHsp90 ATPase activity suggesting that Hsp90 inhibitors could have apotential as new anticancer drugs. Each Hsp90 family member possesses aconserved ATP-binding site at its N-terminal domain, which is found infew other ATP-binding proteins. The weak ATPase activity of Hsp90 isstimulated upon its interaction with various co-chaperone proteins.Several natural compounds such as geldanamycin or radicicol bind at theATP-binding site of Hsp90 inhibiting its ATPase activity. In cellularsystems and in vivo, these drugs upon binding to Hsp90 prevent thefolding of the client proteins, which are then degraded in theproteasome. 17-allylamino-17-demethoxy-geldanamycin (17-AAG), ageldanamycin derivative, is tested in Phase I clinical trials. Initialclinical experiences with 17-AAG have offered preliminary evidence thatconcentrations of the drug associated with activity in pre-clinicalsystems can be achieved in humans with tolerable toxicity, and providedearly evidence of target modulation in at least certain surrogate andtumor compartments. The dose limiting toxicity of 17-AAG is hepatictoxicity. 17-AAG's poor solubility makes it difficult toformulate/administer and its synthesis is difficult (it is generallyobtained by fermentation). Therefore alternative compounds with betterphysicochemical properties and maybe of higher specificity (17-AAGinhibits all these the four Hsp90 paralogs) are needed. WO 2004/072051discloses such alternative compounds, more specifically a series ofisoxazoles derivatives with inhibition properties for heat shockproteins.

5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide is a Hsp90 inhibitors described in WO 2004/072051(Example 78). The compound can be prepared, for instance, as disclosedin such publication.5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide is a poorly water soluble compound, which shows asolubility in water of less than 0.1 mg/ml. The solubility of the freebase in 0.1 N HCl is about 1-2.5 mg/ml. Furthermore, the free base isslightly hygroscopic showing a maximum uptake of 1.8% of water at 80%relative humidity (r.h.) at 25° C.

Surprisingly, new salt forms of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide were found including the hydrochloride, tartrate,phosphate, hemi fumarate and the mesylate salt. These salts and the freebase were found to be crystalline rendering these forms particularlysuitable for pharmaceutical development.

Neither of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate and5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride was found to be hygroscopic.

Furthermore, it was found that5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate is superior compared to the free base as wellas other salts in terms of solubility. Besides the advantage ofexhibiting a high water solubility, the mesylate salt is also suitableto be manufactured reproducibly in a clear acid/base ratio. This findingrenders the mesylate salt particularly suitable for the use in liquidformulations for oral as well as for intravenous formulations.

All of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide salts show higher solubility compared with the freebase.

The solubility of the hydrochloride salt in water is 2.5-5 mg/ml, at pH5 it is approximately 1-2.5 mg/ml and is less than 1 mg/ml in 0.1 N HCl.

The solubility of the tartrate salt in water is more than 2 mg/ml.

The solubility of the phosphate salt in water is approximately 0.5-1.0mg/ml.

The solubility of the hemi fumarate salt in water is approximately0.1-0.25 mg/ml.

The solubility of the mesylate salt at pH 5 is approximately 1-2.5 mg/mland 10-20 mg/ml in 0.1 N HCl. The solubility in water at pH 4 was foundto be 33.5 mg/ml.

In addition to the above, it has been found that5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate can be obtained in the form of hydrates.Modification A is an anhydrous form of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate and forms H_(A) and H_(B) are hydrated forms of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate.

As a further aspect of the present invention, it has been found thatdifferent crystalline forms of the free base and of the mesylate salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide can be obtained.

Finally, it was found that the crystalline form5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride did not change by equilibration insuspension over a range of applied conditions. Lack of polymorphism is abeneficial property for a solid form designated to be developed as amedicament.

Hence, in a first aspect, the present invention relates to new salts of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide, more specifically to5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide tartrate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide phosphate and5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hemi fumarate.

In a second aspect, the present invention relates to new crystallineforms and hydrates of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate.

In a third aspect, the present invention relates to new crystallineforms of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide free base.

Additionally, the present invention relates to the use of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide tartrate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide phosphate and5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hemi fumarate for the manufacture of a medicament forthe treatment of a disorder mediated by Hsp90.

The general terms used hereinbefore and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise indicated.

The expression “disorders mediated by Hsp90” as used herein denotesdisorders, such as tumor diseases, which result from overexpression,activation or dysregulation of Hsp90 or wherein such overexpression,activation or dysregulation play a prominent role.

The expression “free of any other salts” as used herein means thatneither any salts derived from inorganic or organic acids, such assodium chloride, as isotonic agent nor any buffer salts are present. Itwas observed that in the presence of such other salts crystals areformed sooner or later, which renders the solution of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate unstable and which is in particular anunacceptable risk for a formulation designated to be used forintravenous administration.

The expression “chemically stable” as used herein means that5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide does not undergo any chemical reactions, such assolvolysis, especially no hydrolysis, rearrangements or oxidations.

SHORT DESCRIPTION OF THE FIGURES

FIGS. 1 to 13 X-ray diffraction diagrams of crystalline forms of thefree base, the methane sulphonic acid salt, the hydrochloric salt, thetartic acid salt, the phosphoric acid salt and the hemi fumaric acidsalt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide. In the X-ray diagrams, the angle of refraction 2thetaare plotted on the horizontal axis (x-axis) and the relative lineintensity (background-corrected peak intensity) on the vertical(y-axis). X-ray powder diffraction patterns are measured with Cu Kαradiation source (Kα1 radiation, wavelength λ=1.54060 Angström).

FIG. 1 shows the X-ray diffraction diagram of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide free base, form I.

FIG. 2 shows the X-ray diffraction diagram of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide free base, form II.

FIG. 3 shows the X-ray diffraction diagram of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide free base, form III.

FIG. 4 shows the X-ray diffraction diagram of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide free base, form IV.

FIG. 5 shows the X-ray diffraction diagram of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide free base, form V.

FIG. 6 shows the X-ray diffraction diagram of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide free base, form VII.

FIG. 7 shows the X-ray diffraction diagram of form I of the methanesulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide.

FIG. 8 shows the X-ray diffraction diagram of hydrate form H_(A) ofmethane sulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide.

FIG. 9 shows the X-ray diffraction diagram of hydrate form H_(B) ofmethane sulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide.

FIG. 10 shows the X-ray diffraction diagram of the hydrochloric salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide.

FIG. 11 shows the X-ray diffraction diagram of the tartaric acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide.

FIG. 12 shows the X-ray diffraction diagram of the phosphoric acid saltof5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide.

FIG. 13 shows the X-ray diffraction diagram of the hemi fumaric acidsalt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide.

In a preferred embodiment, the form I of the essentially pure methanesulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide shows the X-ray diffraction diagram indicated in FIG. 7.

High preference is also given for the form I of the methane sulphonicacid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide which shows an X-ray diffraction diagram of the typeshown in FIG. 7, with significant peak intensities at 2-theta values:16.7, 19.4 and 22.7±0.2 deg. In samples being free of any additionalmaterials (other crystal forms, hydrates, excepients), it should bepossible to observe the following 2-theta values: 8.0, 11.9, 13.9, 14.2,16.7, 18.5, 19.4, 22.7, 23.3±0.2 deg.

In another preferred embodiment, the form H_(A) of the essentially puremethane sulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide shows the X-ray diffraction diagram indicated in FIG. 8.

High preference is also given for the form H_(A) of the methanesulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide which shows an X-ray diffraction diagram of the typeshown in FIG. 8, with significant peak intensities at 2-theta values:18.1 and 20.5, ±0.2 deg. In samples being free of any additionalmaterials (other crystal forms, anhydrate, excepients), it should bepossible to observe the following 2-theta values: 11.0, 11.6, 14.1,18.1, 20.1, 20.5, 21.1, 21.7, 21.9, 25.8±0.2 deg.

In a other preferred embodiment, the form H_(B) of the essentially puremethane sulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide shows the X-ray diffraction diagram indicated in FIG. 9.

High preference is also given for the form H_(B) of the methanesulphonic acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide which shows an X-ray diffraction diagram of the typeshown in FIG. 9, with significant peak intensity at the 2-theta value of25.4±0.2 deg. In samples being free of any additional materials (othercrystal forms, anhydrate, excepients), it should be possible to observethe following 2-theta values: 6.2, 10.1, 12.6, 14.5, 18.0, 18.5, 19.0,20.4, 20.9, 21.8, 22.2, 25.4±0.2 deg.

In another preferred embodiment, the essentially pure hydrochloric saltof5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide shows the X-ray diffraction diagram indicated in FIG.10.

High preference is also given for the hydrochloric salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide which shows an X-ray diffraction diagram of the typeshown in FIG. 10.

In another preferred embodiment, the essentially pure tataric acid saltof5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide shows the X-ray diffraction diagram indicated in FIG.11.

High preference is also given for the tataric acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide which shows an X-ray diffraction diagram of the typeshown in FIG. 11.

In another preferred embodiment, the essentially pure phosphoric acidsalt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide shows the X-ray diffraction diagram indicated in FIG.12.

High preference is also given for the phosphoric acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide which shows an X-ray diffraction diagram of the typeshown in FIG. 12.

In another preferred embodiment, the essentially pure fumaric acid saltof5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide shows the X-ray diffraction diagram indicated in FIG.13.

High preference is also given for the fumaric acid salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide which shows an X-ray diffraction diagram of the typeshown in FIG. 13.

The term “essentially pure” is understood in the context of the presentinvention to mean especially that at least 90, preferably at least 95,and most preferably at least 99 percent by weight of the crystals of anacid addition salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide are present in the crystal form according to theinvention.

In a further aspect the present invention provides a new formulationcomprising5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate. More specifically, the present inventionprovides an aqueous solution comprising5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate being free of any other salts. Preferably, theaqueous solution has a pH between 3.2 and 5.2.

Preferably, the formulation comprises between around 4.8 and 5.2 mg/mlsolution, more preferably 5.0 mg/ml solution, of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate. The pH of such a solution is around 4.2 andtherefore at the stability maximum of the mesylate salt. This surprisingfinding renders the solution particularly suitable for pharmaceuticaluse. In particular, it allows to use the solution without any otherexcipients, such as buffers, for stabilizing the drug substance.Furthermore, in view of its high stability the solution comprisingbetween around 4.8 and 5.2 mg/ml solution of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate can be subjected to autoclavation (e.g. byheating to about 120° C., for 20 to 40 minutes, e.g. 30 minutes, under apressure of around 2 bar) without causing any color changes ordegradation products.

In one embodiment, the formulation consists of the mesylate salt of anisotonic aqueous solution of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide and 5% w/w of glucose or mannitol. The latter embodimenthas the further advantage that crystal formation can be avoided evenafter longer storage time, e.g. several months. Furthermore, themesylate salt is chemically stable in this formulation. Furthermore, thepH of such a solution prevents Maillard reactions during autoclavationof the 5% glucose solution which would lead to discoloration at neutralpH.

In view of the surprising findings listed above, the present inventionprovides

-   -   a formulation comprising        5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic        acid ethylamide mesylate, which is preferably an aqueous        solution comprising        5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic        acid ethylamide mesylate being free of any other salts, having        preferably a pH between 3.2 and 5.2;    -   in particular a formulation consisting of an isotonic aqueous        solution of the mesylate salt of        5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic        acid ethylamide and 5% w/w of glucose or mannitol;    -   and in particular an aqueous formulation comprising        5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic        acid ethylamide mesylate in a concentration between around 4.8        and 5.2 mg/ml.

The aqueous solution described herein above is particularly useful forintravenous administration of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate.

Furthermore, it was surprisingly found that5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate is light sensitive as solid as well as insolution. Hence, in a further embodiment, the present invention providesfor amber glass containers being filled with the drug substance or theaqueous formulations described herein above.

Furthermore, the present invention provides for a method for treating adisorder mediated by Hsp90 comprising administering to a warm-bloodedanimal in need thereof a therapeutically active amount of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate or5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride.

EXAMPLES

The following Examples illustrate the invention.

Temperatures are measured in degrees celsius (° C.). Unless otherwiseindicated, the reactions take place at room temperature.

Abbreviations

EtOAc ethyl acetate

r.h. relative humidity

RT room temperature

TBME tert-butyl methyl ether

Example 15-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicAcid Ethylamide Hydrochloric Acid Salt

5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (4.19 g, 9 mmol) is dissolved in absolute alcohol (19.7ml) under gentle warming. After stirring for 5 min, 6N aqueoushydrochloric acid (1.5 ml, 9 mmol) is added at RT (about 25° C.). Theclear solution is seeded with a very small amount of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloric acid salt and ethyl acetate (2 ml) is addedwhereupon the mixture becomes turbid slowly. After 1 h EtOAc (5 ml) isadded in portions, and the suspension is stirred at RT overnight. MoreEtOAc (2 ml) is added. The suspension is stirred for about 5 h andfiltered. The filter cake is washed with absolute alcohol/EtOAc 1:1(v/v) and dried for 16 h at 45° C. under reduced pressure to result inthe title compound as a white powder.

Melting point (DSC): 237° C.

Example 25-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicAcid Ethylamide Mesylate—Conditions (A)

A stirred suspension of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (7.76 g, 15 mmol, containing about 9 wt % TBME) inacetone (15 ml) is warmed to 35° C. To the mixture is added a solutionof methanesulfonic acid (1.43 g, 14.85 mmol) in water (3.33 g) over 20min. After 15 min, the solution is warmed to 45° C. and clear filteredinto a warm flask (about 45° C.). The filter is rinsed withacetone/water 9:1 (v/v, 12 ml). The 45° C. warm filtrate is diluted withacetone (53 ml) over 30 min and seeded with5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide methanesulfonic acid salt (4 mg) whereuponcrystallization begins. After 1 h at 45° C. acetone (30 ml) is added.The suspension is stirred at 45° C. for 1 h before it is cooled to 24°C. over 90 min. TBME (20 ml) is added, and the suspension is stirred atabout 24° C. for 15 h. Two more portions of TBME (20 ml each) are addedfollowed by 2 h stirring each time. Thereafter, the suspension is cooledto 0-5° C. and stirred for 3 h before it is filtered. The filter cake iswashed with acetone/tert-butyl methyl ether 3:1 (v/v) and dried underreduced pressure at 50° C. for about 17 h to give 7.18 g (85%) of thetitle compound as a white, crystalline powder.

Melting point: 233° C.

When seeding is left out, the formed crystals are of the same form butmay contain up to 1.5 wt % of acetone.

Example 35-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicAcid Ethylamide Mesylate—Conditions (B)

186.2 mg (0.4 mmol) of5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide methane sulphonic acid are dissolved in 0.75 ml acetone.154 mg of an aqueous solution (25% w/w) of methane sulphonic acid (0.4mmol) are added at RT resulting in a blue solution. After stirring for 2h, 3.5 ml acetone and 0.5 ml TBME are added. After the solution isstirred for further 12 h, further 3.5 ml of TBME is slowly added, andstirring is continued for further 12 h. The obtained emulsion isfiltrated and the obtained solid washed with 0.5 ml TBME. The solid isdried for 16 h at 50° C. at 20 mbar to provide 180 mg of the titlecompound.

Example 4 Hygroscopicity of the Free Base, the Methane Sulphonic AcidSalt and the Hydrochloric Salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicAcid Ethylamide

Hygroscopicity is measured using a Dynamic Water Sorption (DVS)apparatus. Measurements are performed at 25° C. using about 8-10 mg ofsubstance. The r.h. is changed in steps of 10% r.h. as follows:40-0-95-0-40% r.h. The minimum stage time is 60 min, the maximum 180min, if the change in mass of 10 min is less than 0.002%/min, the r.h.is changed to the next step.

Neither of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate and5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride was found to be hygroscopic. The free baseis slightly hygroscopic showing a maximum uptake of 1.8% of water at 80%relative humidity (r.h.) at 25° C.

Example 5 Manufacturing of 50 mg/10 mL Liquid in Ampoule of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicAcid Ethylamide Mesylate

391.95 g of AUY922 mesylate salt (which corresponds to 325 g free base)and 3.25 kg glucose are dissolved in 65 L water for injection. Acolorless to slightly yellowish solution is obtained with a density of1.018±0.005 g/mL at 20° C., an osmolarity of 315±15 mOsm/kg and a pH of4.2±1.0. The solution is filtered through 0.22 μm. 10.5±0.4 mL of thesolution are filled into an amber glass ampoule. The ampoule are sealedunder nitrogen atmosphere and autoclaved for 30 min at 121° C.

Example 6 Dilution of the 50 mg/10 mL Concentrate of Example 5

10 mL of the 50 mg/10 mL ampoule of Example 5 are diluted withcommercially available 250 mL 5% glucose solution and administered via agravimetric infusion line intravenously to the patient.

Example 7 Solubility of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicAcid Ethylamide Mesylate

The solubility of the5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (AUY922) mesylate salt in different aqueous media weredetermined and found to be as follows:

Water (pH of the saturated solution=3.6) 37.5 mg/mL; 0.9% NaCl solution12.8 mg/mL; 5% glucose solution 39 mg/mL; 5% mannitol solution 39 mg/mL;5% glucose solution pH 4 35.6 mg/mL; 5% glucose solution pH 7 37.5mg/mL: Buffer solutions pH 4.0 (acetate buffer) 14.6 mg/mL; pH 5.0(acetate buffer) 3.2 mg/mL; pH 6.0 (phosphate buffer) 0.6 mg/mL; pH 7.0(phosphate buffer) 0.7 mg/mL; pH 8.0 (borate buffer) 1.8 mg/mL.

High solubilities were found in glucose and mannitol solutions. Inbuffered solutions a pH of 4 was leading to highest solubilities.

Example 8 Stress Tests in Solution or Suspensions of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicAcid Ethylamide Mesylate

The stability of the AUY922 mesylate salt in different aqueous mediawere determined under stress conditions (4 weeks at 50° C.) and thefollowing appearance/content recovery/sum of by- and degradationproducts were found:

Water: colorless solution/97.0%/0.32%; 0.9% NaCl: solution colorlesssolution/98.5%/0.34%; 5% glucose solution: colorlesssolution/99.6%/0.34%.

Buffer solutions pH 1.0 (HCl): colorless solution/96.7%/2.83%; pH 2.0(citrate buffer): colorless solution/97.8%/0.58%; pH 3.0 (citratebuffer): colorless solution/99.2%/0.47%; pH 4.0 (acetate buffer):colorless solution/100.5%/0.36%; pH 5.0 (acetate buffer): colorlesssolution/99.1%/0.50%; pH 6.0 (phosphate buffer): colorlesssuspension/96.2%/0.49%; pH 6.8 (phosphate buffer): colorlesssuspension/96.3%/0.79%; pH 8.0 (borate buffer): colorlesssuspension/88.5%/0.26%.

This data demonstrate the stability maximum of the AUY922 mesylate saltat pH 4. Further it was shown that 5% glucose solution resulted in max.stability compared to other aqueous media.

1-3. (canceled)
 4. Method for treating a disorder mediated by Hsp90comprising administering to a warm-blooded animal in need thereof atherapeutically active amount of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide tartrate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide phosphate or5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hemi fumarate.
 5. A salt selected from the groupconsisting of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide tartrate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide phosphate,5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hemi fumarate.
 6. The salt of claim 5, wherein the saltis5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate. 7-9. (canceled)
 10. A crystalline form of thesalt according to claim
 6. 11. The crystalline form according to claim10, which is5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate anhydrate.
 12. Crystalline form I of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate anhydrate according to claim
 11. 13. Thecrystalline form according to claim 12, which shows an X-ray diffractiondiagram of the type shown in FIG. 7, in which the relative peakintensities of each peak do not deviate by more than 10% from therelative peak intensities in the diagram shown in FIG.
 7. 14. Thecrystalline form according to claim 10, which is a hydrate of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate.
 15. Hydrate form H_(A) of the salt accordingto claim 65-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate.
 16. The hydrate form according to claim 15which shows an X-ray diffraction diagram of the type shown in FIG. 8, inwhich the relative peak intensities of each peak do not deviate by morethan 10% from the relative peak intensities in the diagram shown in FIG.8.
 17. Hydrate form H_(B) of the salt according to claim 65-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate.
 18. The hydrate form according to claim 17which shows an X-ray diffraction diagram of the type shown in FIG. 9, inwhich the relative peak intensities of each peak do not deviate by morethan 10% from the relative peak intensities in the diagram shown in FIG.9.
 19. A crystalline form of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride according to claim
 5. 20. A crystallineform according to claim 19 which shows an X-ray diffraction diagram ofthe type shown in FIG. 10, in which the relative peak intensities ofeach peak do not deviate by more than 10% from the relative peakintensities in the diagram shown in FIG.
 10. 21. A crystalline form of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide tartrate according to claim
 5. 22. A crystalline formaccording to claim 21 which shows an X-ray diffraction diagram of thetype shown in FIG. 11, in which the relative peak intensities of eachpeak do not deviate by more than 10% from the relative peak intensitiesin the diagram shown in FIG.
 11. 23. A crystalline form of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide phosphate according to claim
 5. 24. A crystalline formaccording to claim 23 which shows an X-ray diffraction diagram of thetype shown in FIG. 12, in which the relative peak intensities of eachpeak do not deviate by more than 10% from the relative peak intensitiesin the diagram shown in FIG.
 12. 25. A crystalline form of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hemi fumarate according to claim
 5. 26. A crystallineform according to claim 25 which shows an X-ray diffraction diagram ofthe type shown in FIG. 13, in which the relative peak intensities ofeach peak do not deviate by more than 10% from the relative peakintensities in the diagram shown in FIG.
 13. 27. A formulationcomprising5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate according to claim
 6. 28. The formulationaccording to claim 27, which is an aqueous solution comprising5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate being free of any other salts.
 29. Theformulation according to claim 28 having a pH between 3.2 and 5.2 pH.30. A formulation comprising consisting of an isotonic aqueous solutionof the mesylate salt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide according to claim 6 and 5% w/w of glucose.
 31. Aformulation comprising of an isotonic aqueous solution of the mesylatesalt of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide according to claim 6 and 5% w/w of mannitol.
 32. Theformulation according to claim 28 comprising between around 4.8 and 5.2mg/ml of5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide mesylate.
 33. An amber glass container being filled witha formulation according to claim 27.